Many diseases, in particular cancer diseases, are accompanied by a modified gene expression. This may be a mutation of the genes themselves, which leads to an expression of modified proteins or to an inhibition or over-expression of the proteins or enzymes. A modulation of the expression may however also occur by epigenetic modifications, in particular DNA methylation. Such epigenetic modifications do not affect the actual DNA coding sequence. It has been found that DNA methylation processes have substantial implications for the health, and it seems to be clear that knowledge about methylation processes and modifications of the methyl metabolism and DNA methylation are essential for understanding diseases, for the prophylaxis, diagnosis and therapy of diseases.
The precise control of genes, which represent a small part only of the complete genome of mammals, is a question of the regulation under consideration of the fact that the main part of the DNA in the genome is not coding. The presence of such trunk DNA containing introns, repetitive elements and potentially actively transposable elements, requires effective mechanisms for their durable suppression (silencing). Apparently, the methylation of cytosine by S-adenosylmethionine (SAM) dependent DNA methyltransferases, which form 5-methylcytosine, represents such a mechanism for the modification of DNA-protein interactions. Genes can be transcribed by methylation-free promoters, even when adjacent transcribed or not-transcribed regions are widely methylated. This permits the use and regulation of promoters of functional genes, whereas the trunk DNA including the transposable elements is suppressed. Methylation also takes place for the long-term suppression of X-linked genes and may lead to either a reduction or an increase of the degree of transcription, depending on where the methylation in the transcription unit occurs.
Almost all DNA methylation in mammals is restricted to cytosine-guanosine (CpG) dinucleotide palindrome sequences, which are controlled by DNA methyl transferases. CpG dinucleotides are about 1 to 2% of all dinucleotides and are concentrated in so-called CpG islands. A generally accepted definition of CpG islands is an at least 200 bp long DNA region with a CpG content of at least 50%, and wherein the ratio of the number of observed CG dinucleotides and the number of the expected CG dinucleotides is larger than 0.6 (Gardiner-Garden, M., Frommer, M. (1987) J. Mol. Biol. 196, 261-282; incorporated by reference in its entirety). Typically, CpG islands have at least 4 CG dinucleotides in a sequence having a length of 100 base pairs.
If CpG islands are present in promoter areas, they often have a regulatory function over the expression of the respective gene. In general if the CpG island is hypomethylated, expression can take place. Hypermethylation often leads to the suppression of the expression. In the normal state, a tumor suppressor gene is hypomethylated. If a hypermethylation takes place, this will lead to a suppression of the expression of the tumor suppressor gene, which is frequently observed in cancer tissues. In contrast thereto, oncogenes are hypermethylated in healthy tissue, whereas in cancer tissue they are frequently hypomethylated.
Due to the methylation of cytosine, the binding of proteins regulating the transcription is often prevented. This leads to a modification of the gene expression. With regard to cancer for instance, the expression of cell division regulating genes is often affected, e.g. the expression of apoptosis genes is regulated down, whereas the expression of oncogenes is regulated up. The hypermethylation of the DNA also has a long-term influence on the regulation. By the methylation of cytosine, histone de-acetylation proteins can bind by their 5-methylcytosine-specific domain to the DNA. This has as a consequence that histones are de-acetylated, which will lead to a tighter compacting of the DNA. Thereby, regulatory proteins do not have the possibility anymore to bind to the DNA.
For this reason, the accurate determination of DNA methylation levels is very important. A tailored therapy for the respective person can then be determined. Also the effects of a therapy can be monitored. Moreover the accurate detection of DNA levels is also an important tool for developing new approaches for the prevention, diagnosis and treatment of diseases and for the screening for targets.
Therefore a great technical need exists for highly accurate methods for determining the exact methylation level of cytosines, which are methylated in a disease-specific manner. The present invention makes such a method available. The method according to the invention is more powerful than prior art methods.
An overview for detecting 5-methylcytosine may be gathered from the following review article: Fraga F M, Esteller M, Biotechniques 2002 September, 33(3):632, 634, 636-49 (hereby incorporated by reference in its entirety). The most common methods are based on the use of methylation sensitive restriction enzymes capable of differentiating between methylated and unmethylated DNA and on the treatment with bisulfite.
A method, which is based on the use of methylation sensitive restriction enzymes for determining methylation, is the Differential Methylation Hybridization (DMH, [Huang et al, Hum Mol Genet, 8:459-470, 1999; U.S. patent application Ser. No. 09/497,855] both of these cited references are incorporated by reference to their entirety). According to this method, DNA is initially cut with a single non-methylation-specific restriction enzyme, for instance MseI. The obtained fragments are then ligated with linkers. The thus obtained mixture of fragments is then cut with methylation-specific endonucleases, for instance BstUI and/or HpaII, and amplified by means of linker-mediated PCR. The above steps are performed on the one hand with DNA from a diseased tissue and on the other hand with DNA from adjacent healthy tissue of the same tissue type, and the respectively obtained fragments are labeled with different fluorescence dyes. Both fragment solutions are then co-hybridized on a CpG island microarray. The pattern of fluorescent dots visible therein can then be analyzed to find out, for which CpG clones there are differences in the methylation. As a supplement with regard to the technology and methodological details, reference is made to the documents WO03/087774 and U.S. Pat. No. 6,605,432 (both of these cited references are incorporated by reference to their entirety).
But, in general, the use of methylation sensitive enzymes is limited due to the selectivity of the restriction enzyme towards a specific recognition sequence.
Therefore, the treatment with bisulfite, allowing for the specific reaction of bisulfite with cytosine, which, upon subsequent alkaline hydrolysis, is converted to uracil, whereas 5-methylcytosine remains unmodified under these conditions (Shapiro et al. (1970) Nature 227: 1047; hereby incorporated by reference in its entirety)) is currently the most frequently used method for analyzing DNA for 5-methylcytosine. Uracil corresponds to thymine in its base pairing behavior, that is it hybridizes to adenine; whereas 5-methylcytosine does not change its chemical properties under this treatment and therefore still has the base pairing behavior of a cytosine, that is hybridizing with guanine. Consequently, the original DNA is converted in such a manner that 5-methylcytosine, which originally could not be distinguished from cytosine by its hybridization behavior, can now be detected as the only remaining cytosine using “normal” molecular biological techniques, for example, amplification and hybridization or sequencing. All of these techniques are based on base pairing, which can now be fully exploited.
In patent application WO05/038051 (hereby incorporated by reference in its entirety) improvements for the conversion of unmethylated cytosine to uracil by treatment with a bisulfite reagent are described. According to this method the reaction is carried out in the presence of a aliphatic cyclic ether (e.g. dioxane) or in the presence of a n-alkylene glycol compound (e.g. diethylene glycol dimethyl ether). The bisulfite conversion is conducted at a temperature in the range of 0-80° C. with 2 to 5 thermos-pikes (brief incubation at increased temperature of 85-100° C.).
Subsequent to a bisulfite treatment, usually short, specific fragments of a known gene are amplified and either completely sequenced (Olek A, Walter J. (1997) The pre-implantation ontogeny of the H19 methylation imprint. Nat. Genet. 3: 275-6; hereby incorporated by reference in its entirety) or individual cytosine positions are detected by a primer extension reaction (Gonzalgo M L and Jones P A. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25: 2529-31, WO 95/00669; both of these cited references are incorporated by reference in its entirety) or by enzymatic digestion (Xiong Z, Laird P W. (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25: 2535-4; hereby incorporated by reference in its entirety).
Another technique to detect the methylation status is the so-called methylation specific PCR (MSP) (Herman J G, Graff J R, Myohanen S, Nelkin B D and Baylin S B. (1996), Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 93: 9821-6; hereby incorporated by reference in its entirety). The technique is based on the use of primers that differentiate between a methylated and a non-methylated sequence applied after bisulfite treatment of said DNA sequence. The primer either contains a guanine at the position corresponding to the cytosine in which case it will after bisulfite treatment only bind if the position was methylated. Alternatively the primer contains an adenine at the corresponding cytosine position and therefore only binds to said DNA sequence after bisulfite treatment if the cytosine was unmethylated and has hence been altered by the bisulfite treatment so that it hybridizes to adenine. With the use of these primers, amplicons can be produced specifically depending on the methylation status of a certain cytosine and will as such indicate its methylation state.
A further technique is the detection of methylation via a labelled probe, such as used in the so-called Taqman PCR, also known as MethyLight™ (U.S. Pat. No. 6,331,393; hereby incorporated by reference in its entirety). With this technique it became feasible to determine the methylation state of single or of several positions directly during PCR, without having to analyze the PCR products in an additional step.
In addition, detection by hybridization has also been described (Olek et al., WO 99/28498; both of these cited references are incorporated by reference to their entirety).
The quantification of methylation, e.g. a quantitative detection of the DNA methylation level or the amount of methylated or unmethylated DNA, is possible according to the state of the art by several methods (Laird, P. Nat Rev Cancer 2003; 3(4):253-66.; hereby incorporated by reference in its entirety). These methods are usually based on bisulfite treatment and subsequent amplification. In most cases the analysis takes place after the amplification (e.g. Ms-SNuPE, hybridisation on microarrays, hybridisation in solution or direct bisulfite sequencing; for review: Fraga and Esteller 2002, loc. cit.; hereby incorporated by reference in its entirety). However, this “endpoint analysis” leads to several problems; e.g. product inhibition, enzyme instability and decrease of the reaction components, with the result that the amplification does not proceed uniformly. Therefore a correlation between the amount of input DNA and the amount of amplificate does not always exist. As a consequence, the quantification is error-prone (for review: Kains: The PCR plateau phase—towards an understanding of its limitations. Biochem. Biophys. Acta 1494 (2000) 23-27; hereby incorporated by reference in its entirety).
The real time PCR based MethyLight™ technology uses a different approach for a quantification (for review U.S. Pat. No. 6,331,393; hereby incorporated by reference in its entirety). In brief, this method analyses the exponential phase of the amplification instead of the endpoint. Traditionally a threshold cycle number (Ct) is calculated from the fluorescence signal that describes the exponential growth of the amplification (P S Bernard and C T Wittwer, Real-time PCR technology for cancer diagnostics, Clinical Chemistry 48, 2002; hereby incorporated by reference in its entirety). The Ct value is dependent on the starting amount of methylated DNA. By comparing the Ct value of an experimental sample with the Ct value of a standard curve the methylated DNA can be quantified (for review: Trinh et al. 2001, loc. cit.; Lehmann et al.: Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol. 2002 February; 160(2):605-12; both of these cited references are incorporated by reference to their entirety).
There are two commonly used methods to calculate the Ct value. The threshold method selects the cycle when the fluorescence signal exceeds the background fluorescence. The second derivative maximum method selects the cycle when the second derivative of the amplification curve has its maximum. For classical real-time PCR assays both methods produce identical results.
However, both methods do not produce exact results for the quantification of methylation via MethyLight™ assays. The MethyLight™ technology normally uses a methylation specific amplification (by methylation specific primers or blockers, sometimes methylation unspecific primers are used) combined with a methylation specific probe (for review: Trinh et al., 2001, loc. cit.; hereby incorporated by reference in its entirety). The methylation specific probe results in fluorescence signals from only a part of the generated amplificates depending on the methylation status of the CpG positions covered by the probe. This results in amplification curves that are downscaled compared to curves from completely methylated template DNA. These downscaled curves are the reason that both analysis methods generate incorrect results.
The threshold method assumes that all curves are in their exponential growth phase when exceeding the threshold. However, for samples with low proportions of DNA that is methylated at the probe (especially common in cancer diagnostics) this is not true. Amplification curves are already in the plateau phase and Ct estimation will be wrong.
The second derivative maximum method is independent from the overall intensity of the amplification curve. It only takes the shape into account which corresponds to a quantification of DNA that is methylated at the priming sites. The information generated by the methylation specific probe—represented by the signal intensity—is not used.
An improved method is the MethyLight™ ALGO™ method (EP 04090255.3; hereby incorporated by reference in its entirety), which is based on the MethyLight™ technology. According to this improved method, the degree of methylation is calculated from the signal intensities of probes using different algorithms.
A further method is the so-called QM™ assay (for review PCT/EP2005/003793, hereby incorporated by reference in its entirety), which is also based on real-time PCR. According to this method, a non-methylation-specific, conversion-specific amplification of the target DNA is produced. The amplificates are detected by means of the hybridization of two different methylation-specific real-time PCR probes. One of the probes is specific for the methylated state, while the other probe is specific for the unmethylated state. The two probes are labelled with different fluorescent dyes. The quantification of the degree of methylation can be carried out during specific PCR cycles by employing the ratio of signal intensities of the two probes. Alternatively, the Ct values of two fluorescent channels can also be drawn on for the quantification of the methylation. In both cases, quantification of the degree of methylation is possible without the necessity of determining the absolute DNA quantity. A simultaneous amplification of a reference gene or a determination of PMR values is thus not necessary. In addition, the method supplies reliable values for both large and small DNA quantities as well as for high and low degrees of methylation.
The third preferred method for quantitative detection of DNA methylation is the so-called restriction assay, also known as Mest evaluation (PCT/DE205/001109; hereby incorporated by reference in its entirety). In brief, according to this method, DNA is digested with at least one methylation-specific restriction enzyme. After this, the digested DNA is subjected to real time PCR amplification. But amplificates are only amplified from said DNA if the DNA was not previously cut by the methylation-specific enzyme or enzymes within the sequence of the amplificate. The percentage of methylated DNA is then deduced by comparison of the signal intensity obtained for the sequence of interest with that of a reference sample.
According to the said methods, a quantification of methylation levels is possible. But recent studies have shown, that they only have a limited accuracy making a precise characterization of samples very difficult. Therefore the differentiation, grading, and staging of diseased tissue is impaired and therefore also the diagnosis of proliferative disorders or predisposition to those.
Because of that, it is the technical object of the invention to provide a quantitative method for DNA methylation analysis which has a higher accuracy than the known methods. Consequently, a liable differentiation, grading, and staging of diseased tissue and therefore also the diagnosis of proliferative disorders or predisposition to those is enabled.
Very surprisingly, this technical need can be fulfilled by a simple approach according to the invention. The present invention addresses the problem, heretofore unrecognized, that the majority of biological samples isolated from a patient are a heterogenous mixture of a plurality of pathologically cell types e.g. healthy tissue and diseased tissue. The method of the invention enables the quantification of selected tissue or cellular type(s) within said heterogenous biological sample. Said method is particularly useful in the field of pathology wherein a biological sample from a patient is often a heterogeneous mixture of healthy and sick cells. By enabling the quantification of the amount of healthy and sick cells within a sample the invention assists in the quantification of disease markers within said sample.